Glucokinase activator compositions for the treatment of diabetes

ABSTRACT

The present invention relates to the use of liver-selective glucokinase activators and a GLP1 analog in restoring insulin sensitivity and treating type II diabetes, including reducing body weight in subjects undergoing type II diabetes treatment.

SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronictext file named “0378-214-US_Sequence_Listing.txt”, having a size inbytes of 846, and created on Jul. 10, 2018. The information contained inthis electronic file is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical compositions comprising{2-[3-cyclohexyl-3-(trans-4-propoxy-cyclohexyl)-ureido]-thiazol-5-ylsulfanyl}-aceticacid (FRI-1) in combination with an anti-diabetic drug selected from thegroup consisting of metformin, sitagliptin or exenatide. The presentinvention also relates to the use of the pharmaceutical compositions inrestoring insulin sensitivity and treating type II diabetes, includingreducing body weight in subjects undergoing type II diabetes treatment.

BACKGROUND OF THE INVENTION

Diabetes is a disorder characterized by impaired glucose metabolismmanifested by an elevated glucose level in subjects. There are two formsof diabetes based on the underlying defects of the disease. Type Idiabetes arises when subjects lack pancreatic β-cells producing insulin,the hormone that regulates glucose utilization. Type II diabetes ariseswhen subjects have impaired β-cell function, including otherabnormalities. In type II diabetic subjects, plasma insulin levels maybe the same or even elevated compared to non-diabetic subjects. Suchplasma insulin levels, while elevated, lead to impairedinsulin-stimulated glucose uptake in muscles. Also, insulin-resistantadipocytes have diminished capacity to mobilize lipids andtriglycerides. Consequently, an increase in circulating glucose andlipids is seen, leading to the metabolic abnormalities often associatedwith type II diabetes.

Type I subjects are currently treated with insulin. While the majorityof type II subjects are treated with sulfonylureas or metformin, thesesubjects gradually lose the ability to respond to monotherapy, thusrequiring treatment with multiple drugs.

An option for normalizing blood glucose levels is the use of combinationtherapies. For example, an FDA approved combination treatment of type IIdiabetes is the use of dipeptidyl peptidase-IV (DPP-IV) inhibitors withmetformin. Another combination treatment currently under investigationuses glucokinase (“GK”) activators in combination with metformin and isdescribed in WO11/149945.

The long-term efficacy of therapies for type II diabetes is limited bythe risk of developing side effects, for example, hypoglycemia andweight gain in subjects undergoing treatment. Accordingly, there is aneed to find better options for treating type II diabetes, which includerestoring insulin sensitivity and/or controlling weight gain in subjectsundergoing such treatment.

SUMMARY OF THE INVENTION

The present invention provides for pharmaceutical compositionscomprising one or more glucokinase activators and one or moreanti-diabetic drugs, as well as their use in treating type 2 diabetesand related disorders.

In one aspect, the present invention provides for a pharmaceuticalcomposition comprising a glucokinase activator, or a pharmaceuticallyacceptable salt thereof, an anti-diabetic drug selected from the groupconsisting of a DPP-IV inhibitor, a GLP-1 analog, or a pharmaceuticallyacceptable salt thereof and at least one pharmaceutically acceptablecarrier, excipient, diluent or mixture thereof. In some embodiments, theglucokinase activator, the anti-diabetic drug, or both are present insuboptimal amounts. The glucokinase activator can be a liver-selectiveglucokinase activator. In some embodiments, the glucokinase activatorcan be{2-[3-cyclohexyl-3-(trans-4-propoxy-cyclohexyl)-ureido]-thiazol-5-ylsulfanyl}-aceticacid or a pharmaceutically acceptable salt thereof. In otherembodiments, the anti-diabetic drug is sitagliptin, exenatide, or apharmaceutically acceptable salt thereof.

In another aspect, the present invention provides for a method oftreating type II diabetes comprising administering to a subject apharmaceutical composition comprising a glucokinase activator or apharmaceutically acceptable salt thereof and an anti-diabetic drugselected from the group consisting of a DPP-IV inhibitor and a GLP-1analog or a pharmaceutically acceptable salt thereof. In someembodiments, the glucokinase activator, the anti-diabetic drug, or bothare present in suboptimal amounts. The glucokinase activator can be aliver-selective glucokinase activator. In some embodiments, theglucokinase activator can be{2-[3-cyclohexyl-3-(trans-4-propoxy-cyclohexyl)-ureido]-thiazol-5-ylsulfanyl}-aceticacid or a pharmaceutically acceptable salt thereof. In otherembodiments, the anti-diabetic drug is sitagliptin, exenatide, or apharmaceutically acceptable salt thereof.

In another aspect, the present invention provides for a method ofimproving glycemic control comprising administering to a subject apharmaceutical composition comprising a glucokinase activator or apharmaceutically acceptable salt thereof and an anti-diabetic drugselected from the group consisting of DPP-IV inhibitor and a GLP-1analog or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides for a method oftreating a condition in a subject comprising administering to apharmaceutical composition comprising a glucokinase activator or apharmaceutically acceptable salt thereof and an anti-diabetic drugselected from the group consisting of DPP-IV inhibitor and a GLP-1analog or a pharmaceutically acceptable salt thereof, wherein thecondition is selected from the group consisting of a metabolic disorder,glucose intolerance, prediabetic state, insulin resistance,hyperglycemia, impaired glucose tolerance (IGT), Syndrome X, impairedfasting glucose (IFG), type I diabetes, dyslipidemia, hyperlipidemia,hyperlipoproteinemia, hypertension, osteoporosis, non-alcoholic fattyliver disease (NAFLD), complications resulting from or associated withdiabetes, cardiovascular disease, and obesity.

In another aspect, the present invention provides for a method ofnormalizing or lowering blood glucose; delaying IGT to type II diabetes;delaying the progression of non-insulin-requiring type II diabetes toinsulin-requiring type II diabetes; lowering of food intake; regulatingappetite; regulating feeding behavior; enhancing secretion ofenteroincretins; improving glucose tolerance; reducing fasting plasmaglucose; reducing postprandial plasma glucose; reducing glycosylatedhemoglobin HbA1c; slowing progression of, delaying or treatingcomplications of diabetes; reducing weight or preventing an increase ofweight or facilitating a reduction of weight; treating the degenerationof pancreatic beta cells; improving and/or restoring functionality ofpancreatic beta cells; stimulating and/or restoring functionality ofpancreatic insulin secretion; enhancing phosphorylation of glucose;maintaining insulin sensitivity; improving insulin sensitivity; treatinghyperinsulinemia; or treating insulin resistance comprisingadministering to a subject a pharmaceutically acceptable salt thereofand an anti-diabetic drug selected from the group consisting of DPP-IVinhibitor and a GLP-1 analog or a pharmaceutically acceptable saltthereof.

Other features and aspects of the present invention are also describedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts synergistic effects of a combination of FRI-1 andmetformin on body weight reduction in male ob/ob mice.

FIG. 2 depicts pharmacological effects of a combination of FRI-1 andmetformin on plasma glucose and insulin in male ob/ob mice.

FIG. 3 depicts synergistic effects of a combination of FRI-1 andmetformin on increasing insulin sensitivity as measured by the insulinsensitivity index (ISI).

FIG. 4 depicts synergistic effects of a combination of FRI-1 andsitagliptin on body weight reduction in female diet-induced obese rats.

FIG. 5 depicts pharmacological effects of a combination of FRI-1 andexenatide on plasma glucose and insulin in male ob/ob mice.

FIG. 6 depicts synergistic effects of a combination of FRI-1 andexenatide on increasing insulin sensitivity as measured by the insulinsensitivity index.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “GK activator” refers to a compound that sensitizes theglucokinase (GK) sensor system. GK is an enzyme that belongs to thefamily of hexokinases, which catalyze the first step in the metabolismof glucose, i.e., conversion of glucose to glucose-6-phosphate. GKfunctions as a glucose sensor in the pancreas and liver. In oneembodiment, the GK activator is a liver selective activator that doesnot increase insulin secretion by the pancreas in presence of glucose.Exemplary GK activators include FRI-1 or those disclosed in WO05/066145.In one embodiment, a GK activator is FRI-1.

The term “FRI-1” is represented by the chemical name{2-[3-cyclohexyl-3-(trans-4-propoxy-cyclohexyl)-ureido]-thiazol-5-ylsulfanyl}-aceticacid. As used herein, FRI-1 is not limited to the free acid, but alsoincludes pharmaceutically acceptable salts of FRI-1. In one embodiment,FRI-1 is a free acid.

The term “anti-diabetic drug” refers to agents found in the literature.As used herein, anti-diabetic drugs include pharmaceutically acceptablesalts, pro-drugs, and pharmaceutically acceptable salts of pro-drugs ofanti-diabetic drugs. Anti-diabetic drugs for example fall within thecategories of insulin, insulin sensitizing active agents, active agentsthat enhance the production of insulin, sulfonamides, biguanidinederivatives and α-glucosidase inhibitors. Insulin, for example, is humaninsulin prepared by recombinant technology. The insulin sensitizingactive agents enhance the effect of insulin, including, for example,PPAR (peroxisome proliferator-activated receptor) α-agonists, includingthiazolidinedione derivatives, such as pioglitazone, troglitazone,ciglitazone, rivoglitazone, rosiglitazone or other 2,4-thiazolidinedionederivatives. Active agents that enhance the production of insulininclude, for example, DPP-IV inhibitors, such as sitagliptin,vildagliptin, saxagliptin, linagliptin, dutogliptin, gemigliptin oralogliptin; GLP-1 analogs, such as exenatide, liraglutide, taspoglutide,albiglutide, or lixisenatide; and ATP-sensitive potassium channelmodulators, such as mitiglinide, repaglinide or nateglinide.Sulfonamides include, for example, sulfonylurea derivatives, such astolbutamide, chlorpropamide, tolazamide, acetohexamide, glipizide,gliclazide, glimepiride, gliquidone, glibornuride, glisoxepid,glibenclamide, glisentide, glisolamide, glybuzole, or glyclopyramide.Biguanidine derivatives include, for example, metformin, buformin, orphenformin. α-glucosidase inhibitors include, for example, miglitol,acarbose or voglibose. In one embodiment, an anti-diabetic drug may beincluded in any dosage form, e.g., oral, inhaled or an injectable dosageform. In another embodiment, an anti-diabetic drug is in an oral dosageform.

The term “metformin” is represented by the chemical name,N,N-dimethylimidodicarbonimidic diamide. As used herein, metformin isnot limited to the free base, but also includes pharmaceuticallyacceptable salts of metformin. In one embodiment, metformin is metforminhydrochloride.

The term “DPP-IV inhibitor” means a compound that inhibits the action ofdipeptidyl peptidase IV from cleaving dipeptides located at N-terminalportions of proteins having either an N-terminal proline or alanineresidue. In one embodiment, a DPP-IV inhibitor is sitagliptin.

The term “sitagliptin” is represented by the chemical name,(3R)-3-amino-1-[3-(trifluoromethyl)-5,6,7,8-tetrahydro-5H-[1,2,4-]triazolo[4,3-a]pyrazin-7-yl]-4-2,4,5-trifluorophenyl)butan-1-one.As used herein, sitagliptin is not limited to the free base, but alsoincludes pharmaceutically acceptable salts of sitagliptin, and isomersof sitagliptin. In one embodiment, sitagliptin is sitagliptin phosphate.

The term “GLP-1 analog” means a glucagon-like peptide-1 compound havingsequence similarity of about 30% to about 90%, or about 40% to about 75%to glucagon like peptide-1 (also referred to as pro-glucagon). GLP-1 hasan insulinotropic effect, stimulating insulin secretion from pancreaticbeta cells. In one embodiment, a GLP-1 analog is exenatide.

The term “exenatide” represents a 39-amino acid peptide with sequenceH-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 (SEQ. ID. No: 1). As used herein, exenatide is notlimited to the free base, but also includes pharmaceutically acceptablesalts of exenatide. In one embodiment, exenatide is a free base.

The term “pharmaceutically acceptable salt” represents those salts whichare suitable for use in contact with the tissues of humans and loweranimals without undue toxicity, irritation, allergic response and thelike, and are commensurate with a reasonable benefit/risk ratio.Pharmaceutically acceptable salts include those obtained by reacting themain compound, functioning as a base with an inorganic or organic acidto form a salt, for example, salts of hydrochloric acid, sulfuric acid,phosphoric acid, methane sulfonic acid, camphor sulfonic acid, oxalicacid, maleic acid, succinic acid, citric acid, formic acid, hydrobromicacid, benzoic acid, tartaric acid, fumaric acid, salicylic acid,mandelic acid, and carbonic acid. Pharmaceutically acceptable salts alsoinclude those in which the main compound functions as an acid and isreacted with an appropriate base to form, e.g., sodium, potassium,calcium, magnesium, ammonium, and choline salts. Those skilled in theart will further recognize that acid addition salts of the claimedcompounds may be prepared by reaction of the compounds with theappropriate inorganic or organic acid via any of a number of knownmethods. Alternatively, alkali and alkaline earth metal salts can beprepared by reacting the compounds of the invention with the appropriatebase via a variety of known methods.

The following are further examples of acid salts that can be obtained byreaction with inorganic or organic acids: acetates, DIPEAtes, alginates,citrates, aspartates, benzoates, benzenesulfonates, bisulfates,butyrates, camphorates, digluconates, cyclopentanepropionates, dodecylsulfates, ethanesulfonates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, fumarates, hydrobromides,hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates,methanesulfonates, nicotinates, 2-naphthalenesulfonates, oxalates,palmoates, pectinates, persulfates, 3-phenylpropionates, picrates,pivalates, propionates, succinates, tartrates, thiocyanates, tosylates,mesylates and undecanoates. In one embodiment, the pharmaceuticallyacceptable salt can be a hydrochloride, a hydrobromide, a hydroformate,or a maleate salt.

The term “in combination” means a pharmaceutical composition that placesno limit, i.e., method, form, etc., on the administering of a compoundin combination with another compound. For example, in some embodiments,FRI-1 and an anti-diabetic drug are administered together in a singledosage form, e.g., fixed dose combination. In other embodiments, FRI-1and an anti-diabetic drug are administered in separate, discrete dosageforms e.g., one may be an oral preparation and the other may be aninhaled dose form, or as same dosage forms, or in separate containers,e.g., blisters. In another embodiment, both FRI-1 and an anti-diabeticdrug are administered at the same time or are taken sequentiallyadministered about 5 minutes apart, or about 15 minutes apart, or about30 minutes apart, or about 1 hour apart, or about 2 hours apart, orabout 4 hours apart, or about 8 hours apart, or about 12 hours apart, orabout 24 hours apart, wherein FRI-1 is administered earlier than theanti-diabetic drug, or vice versa.

The term “treating or treatment” means to manage or control a disease,condition or disorder. This includes relieving, alleviating,ameliorating, delaying, reducing, reversing, or improving a disease,disorder or condition or at least one symptom thereof; delaying theonset of a disease, disorder, or condition; or delaying the recurrenceof a disease, disorder, or condition, or characteristic symptomsthereof, depending on the nature of the disease, disorder, or conditionand its characteristic symptoms.

The term “subject” means animals, including both males and females. Inone embodiment, subject means mammals. In another embodiment, subjectmeans humans.

The term “therapeutic effect” means an amount of an active ingredient(e.g., GK activator or an anti-diabetic drug) that elicits thebiological or medicinal response in a tissue, system, or subject that isbeing sought by a researcher, veterinarian, medical doctor, patient orother clinician, which includes reduction or alleviation of the symptomsof the disease being treated.

The term “glycemic control” means the management of diabetes as measuredby the diagnostic parameters or HbA1c and/or FPG. Subjects withinadequate or insufficient glycemic control include subjects having aHbA1c value from about 7.5% to about 15% at baseline, from about 8% toabout 13% at baseline, and from about 9% to about 12% at baseline. Inone embodiment, subjects with inadequate glycemic control includesubjects having a HbA1c value from 7.5% to about 10% at baseline despitetreatment with metformin.

As used herein, the term “consisting of” is not an absolute restrictionand includes unrecited components. Typically, the unrecited componentsinclude an impurity ordinarily associated therewith or a componentunrelated to the invention.

II. Pharmaceutical Compositions

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising a GK activator in combination with ananti-diabetic drug, and at least one pharmaceutically acceptablecarrier, diluent or excipient. In one embodiment, the pharmaceuticallyacceptable carriers are present in two discrete dosage forms, one dosageform comprising a GK activator and the other dosage form comprising ananti-diabetic drug. In another embodiment, the active ingredients of thecombination are combined in a single dosage form. In yet anotherembodiment, the dosage form is intended for oral use. In anotherembodiment, the pharmaceutical composition consists of a GK activatorand an anti-diabetic drug.

Compositions intended for oral use may be prepared according to anyknown method, and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavoringagents, coloring agents, and preserving agents in order to providepharmaceutically elegant and palatable preparations. Tablets may containthe active ingredient in admixture with non-toxicpharmaceutically-acceptable excipients which are suitable for themanufacture of tablets. These excipients may be for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example corn starch or alginic acid; binding agents, for example,starch, gelatin or acacia; and lubricating agents, for example,magnesium stearate, stearic acid or talc.

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising FRI-1 or a pharmaceutically acceptable saltthereof in combination with an anti-diabetic drug wherein theanti-diabetic drug is selected from the group consisting of metformin,sitagliptin and exenatide, or a pharmaceutically acceptable saltthereof.

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising FRI-1 or a pharmaceutically acceptable saltthereof in combination with metformin or a pharmaceutically acceptablesalt thereof.

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising FRI-1 or a pharmaceutically acceptable saltthereof in combination with sitagliptin or a pharmaceutically acceptablesalt thereof.

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising FRI-1 or a pharmaceutically acceptable saltthereof in combination with exenatide or a pharmaceutically acceptablesalt thereof.

We have surprisingly found that the administration of FRI-1 incombination with metformin, sitagliptin or exenatide is synergistic inthe context of modifying glucose metabolism in subjects in need of suchmodification. In another embodiment, we have surprisingly found that theadministration of FRI-1 in combination with sitagliptin is synergisticin controlling body weight in subjects having impaired glucosemetabolism. Further, we have surprisingly found that the administrationof FRI-1 in combination with exenatide is synergistic in the context ofimproving insulin sensitivity in subjects with impaired glucosemetabolism. Additionally we have surprisingly found the administrationof FRI-1 in combination with metformin is synergistic both in thecontext of controlling body weight in subjects with impaired glucosemetabolism as well as improving insulin sensitivity in subjects withimproved glucose metabolism. In one embodiment, the present inventionprovides for small doses of either FRI-1 or an anti-diabetic drug orboth, wherein the small doses are such that they are less than theoptimum dose of either FRI-1 or anti-diabetic drug for a therapeuticeffect. In another embodiment, small doses of either active ingredientof the present invention are administered simultaneously or sequentiallyin any order.

Pharmaceutical compositions can be prepared by methods known by one ofordinary skill in the art, including the selection of pharmaceuticallyacceptable ingredients.

In one embodiment, the present invention provides for a pharmaceuticalcomposition that is an oral dosage form administered as discrete solidunits, e.g., capsules, tablets, pills, powders, granules etc. Oraldosage forms can include any pharmaceutical carrier, diluents (such assucrose, mannitol, lactose, starches) or excipients known in the art,including but not limited to suspending agents, solubilizers, bufferingagents, binders, disintegrants, preservatives, colorants, flavorants,lubricants may be used. In some embodiments, the present inventionprovides an oral dosage form for either or both of GK activator andanti-diabetic drug.

In another embodiment, the present invention provides for apharmaceutical composition that is an oral dosage administered as aliquid form. Exemplary liquid oral dosage forms are aqueous andnon-aqueous solutions, emulsions, suspensions, syrups, and elixirs. Suchdosage forms can also contain suitable inert diluents known in the artsuch as water and suitable excipients known in the art such aspreservatives, wetting agents, sweeteners, flavorants, as well as agentsfor emulsifying and/or suspending the compounds of the invention.

In yet another embodiment, the present invention provides for apharmaceutical composition that is an injectable dosage form, forexample, intravenously, in the form of an isotonic sterile solution. Inyet another embodiment, the present invention provides an injectabledosage form for either or both of GK activator and anti-diabetic drug.

In another embodiment, the present invention provides for apharmaceutical composition that is an inhalable dosage form, for examplein the form of a powder (e.g., micronized) or in the form of atomizedsolutions or suspensions. In yet another embodiment, the presentinvention provides an inhalable dosage form for either or both of GKactivator and anti-diabetic drug.

In some embodiments, the pharmaceutical composition is a single dosageform comprising one or more GK activators and one or more anti-diabeticdrugs. In other embodiments, two or more dosage forms are provided,where at least one dosage form comprises one or more GK activators andat least one other dosage form comprises one or more anti-diabetic drug.

III. Dosage Quantities

The dosage of the pharmaceutical composition of the present inventionwill vary depending on the symptoms, the treatment desired, age and bodyweight of the subject, the nature and severity of the disorder to betreated, the route of administration and pharmacokinetics of the activeingredients. The frequency of the dose indicated will also vary with thetreatment desired and the disorder indicated.

In one embodiment, a GK activator is administered in combination with ananti-diabetic drug in an amount sufficient to achieve a therapeuticeffect. The dosage range for a GK activator ranges from about 1 mg toabout 1000 mg per day. In other embodiments, the amount for a GKactivator ranges from about 5 mg to about 900 mg per day, or about 10 mgto about 800 mg per day, or about 50 mg to about 700 mg per day, orabout 150 mg to about 500 mg per day, or about 200 mg to about 400 mgper day. The dosage range for an anti-diabetic drug ranges from about0.1 μg to about 2000 mg per day. In other embodiments, the amount for ananti-diabetic drug ranges from about 0.5 μg to about 1000 mg per day, orabout 1 μg to about 750 mg per day, or about 5 μg to about 500 mg perday, or about 20 μg to about 250 mg per day, or about 100 μg to about100 mg per day, or about 500 μg to about 10 mg per day, or about 1 mg toabout 5 mg per day.

In yet other embodiments, the dosage of FRI-1 is about 0.05 mg/kg ofbody weight per day, or about 0.1 mg/kg of body weight per day, or about0.3 mg/kg of body weight per day, or about 1 mg/kg of body weight perday or about 5 mg/kg of body weight per day, or about 25 mg/kg of bodyweight per day, or about 100 mg/kg of body weight per day, or about 200mg/kg of body weight per day or about 500 mg/kg of body weight per day;and the dosage for anti-diabetic drugs, wherein the anti-diabetic drugis selected from the group consisting of metformin, sitagliptin, andexenatide, is about 0.005 mg/kg of body weight per day, or about 0.01mg/kg of body weight per day, or about 0.05 mg/kg of body weight perday, or about 0.1 mg/kg of body weight per day, or about 0.3 mg/kg ofbody weight per day, or about 1 mg/kg of body weight per day or about 5mg/kg of body weight per day, or about 25 mg/kg of body weight per day,or about 100 mg/kg of body weight per day, or about 200 mg/kg of bodyweight per day or about 500 mg/kg of body weight per day. One skilled inthe art will appreciate that the administered doses can be converted toa human equivalent dose.

Metformin is known to one skilled in the art and may be administered asa monotherapy in an amount of 500 mg per day to 2550 mg per day,typically in the form of 500 mg, 850 mg and 1000 mg tablets. In smaller,suboptimal doses, metformin may offer no or negligible therapeuticbenefits when administered as a monotherapy. Metformin can beadministered in suboptimal doses, in combination with a GK activator,and provide a therapeutic benefit (for example, less than 500 mg daily).Metformin can also be administered in optimal doses, in combination witha GK activator, and provide a synergistic therapeutic benefit. In someembodiments, the administered dose of metformin can be from about 100 mgto about 2600 mg per day, or from about 250 mg to about 2500 mg per day,or from about 500 mg to about 1500 mg per day, or from about 250 mg toabout 1000 mg per day, or from about 350 mg to about 850 mg per day, orfrom about 400 mg to about 750 mg per day. In other embodiments, theadministered dose of metformin is less than 500 mg per day, for example,100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg,325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg or 475 mg per day.

Sitagliptin is known to one skilled in the art and may be administeredas a monotherapy in an amount of 100 mg per day, while in patients withmoderate, severe and end stage renal disease the administered amount maybe 25 mg or 50 mg per day. In smaller, suboptimal doses, sitagliptin mayoffer no or negligible therapeutic benefits when administered as amonotherapy. Sitagliptin can be administered in suboptimal doses, incombination with a GK activator, and provide a therapeutic benefit (forexample, less than 100 mg daily is a suboptimal amount for patientswithout renal disease, and less than 25 mg daily is a suboptimal amountfor patients with renal disease). Sitagliptin can also be administeredin optimal doses, in combination with a GK activator, and provide asynergistic therapeutic benefit. In some embodiments, the administereddose of sitagliptin ranges from about 0.1 mg to about 500 mg per dayadministered orally. In other embodiments, the amount of sitagliptinranges from about 0.25 mg to about 400 mg per day, or from about 0.5 mgto about 250 mg per day, or from about 1 mg to about 100 mg per day, orfrom about 5 mg to about 50 mg per day, or from about 10 mg to about 25mg per day. In other embodiments, the amount of sitagliptin is less than100 mg per day, for example, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg,55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg or 95 mg per day.In yet other embodiments, the amount of sitagliptin is less than about25 mg per day, for example, about 0.1 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg or 22.5 mg perday.

Exenatide is known to one skilled in the art and can be administered asa monotherapy in an amount of 10 μg to 20 μg per day or 2 mg per week.In smaller, suboptimal doses, exenatide may offer no or negligibletherapeutic benefits when administered as a monotherapy. Exenatide canbe administered in suboptimal doses, in combination with a GK activator,and provide a therapeutic benefit (for example, less than 500 mg daily).Exenatide can also be administered in optimal doses, in combination witha GK activator, and provide a synergistic therapeutic benefit. In oneembodiment, the dose of exenatide is 0.1 μg to about 100 μg per dayadministered subcutaneously. In other embodiments, the amount ofexenatide ranges from about 0.25 μg to about 75 μg per day, from about0.5 μg to about 50 μg per day, from about 1 μg to about 25 μg per day,or from 5 μg to about 10 μg per day. In other embodiments, the amount ofexenatide is less than about 10 μg per day, for example, about 0.25 μg,0.5 μg, 1 μg, 1.5 μg, 2 μg, 2.5 μg, 3 μg, 3.5 μg, 4 μg, 4.5 μg, 5 μg,5.5 μg, 6 μg, 6.5 μg, 7 μg, 7.5 μg, 8 μg, 8.5 μg, 9 μg or 9.5 μg perday.

IV. Methods of Treatment

In one embodiment, the present invention provides for treating subjectscomprising administering to a subject in need thereof a pharmaceuticalcomposition comprising a GK activator in combination with ananti-diabetic drug for the following methods:

(a) treating type I diabetes and/or type II diabetes.

(b) normalizing or lowering blood glucose levels;

(c) improving glucose tolerance;

(d) improving glycemic control;

(e) reducing fasting plasma glucose;

(f) reducing postprandial plasma glucose

(g) reducing glycosylated hemoglobin HbA1c;

(h) slowing progression of, delaying or treating complications ofdiabetes, e.g., diabetic nephropathy, retinopathy, neuropathy orcardiovascular disease;

(i) reducing weight or preventing an increase of weight or facilitatinga reduction of weight;

(j) treating the degeneration of pancreatic beta cells;

(k) improving and/or restoring functionality of pancreatic beta cells;

(l) stimulating and/or restoring functionality of pancreatic insulinsecretion;

(m) enhancing phosphorylation of glucose; or

(n) maintaining and/or improving insulin sensitivity; and/or treatinghyperinsulinemia and/or insulin resistance.

In one embodiment, the present invention provides for normalizing bloodglucose levels and improving glucose tolerance in a subject byadministering to the subject a pharmaceutical composition comprisingFRI-1 or a pharmaceutically acceptable salt thereof in combination withan anti-diabetic drug, wherein the anti-diabetic drug is selected fromthe group consisting of metformin, sitagliptin and exenatide or apharmaceutically acceptable salt thereof.

In another embodiment, the present invention provides for improvingglycemic control; and/or for reducing fasting plasma glucose, reducingpostprandial plasma glucose and/or reducing glycosylated hemoglobinHbA1c in a subject by administering to the subject a pharmaceuticalcomposition comprising FRI-1 or a pharmaceutically acceptable saltthereof in combination with an anti-diabetic drug, wherein theanti-diabetic drug is selected from the group consisting of metformin,sitagliptin and exenatide or a pharmaceutically acceptable salt thereof.In an embodiment, a method may reduce the amount of HbA1c in a subjectin need thereof by at least 0.1 of a percentage point, or 0.2 of apercentage point, or 0.3 of a percentage point, or 0.4 of a percentagepoint, or 0.5 of a percentage point, or 0.6 of a percentage point, or0.7 of a percentage point, or 0.8 of a percentage point, or 0.9 of apercentage point, or one percentage point. In still other embodiments,the method may reduce the level of HbA1c in a subject in need thereof toless than 7%. In other embodiments, the level of HbA1c may be reduced toa level between 5 and 6.5%.

In another embodiment, the present invention provides for slowingprogression of, delaying or treating complications in a subject (e.g.,diabetic nephropathy, retinopathy, neuropathy or cardiovascular disease)by administering to the subject a pharmaceutical composition comprisingFRI-1 or a pharmaceutically acceptable salt thereof in combination withan anti-diabetic drug, wherein the anti-diabetic drug is selected fromthe group consisting of metformin, sitagliptin and exenatide or apharmaceutically acceptable salt thereof.

In yet another embodiment, the present invention provides for reducingweight or preventing an increase of weight or facilitating a reductionof weight in a subject by administering to the subject a pharmaceuticalcomposition comprising FRI-1 or a pharmaceutically acceptable saltthereof in combination with an anti-diabetic drug, wherein theanti-diabetic drug is selected from the group consisting of metformin,sitagliptin and exenatide or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention provides for treating thedegeneration of pancreatic beta cells; and/or improving and/or restoringfunctionality of pancreatic beta cells; and/or stimulating and/orrestoring functionality of pancreatic insulin secretion in a subject byadministering to the subject a pharmaceutical composition comprisingFRI-1 or a pharmaceutically acceptable salt thereof in combination withan anti-diabetic drug, wherein the anti-diabetic drug is selected fromthe group consisting of metformin, sitagliptin and exenatide or apharmaceutically acceptable salt thereof.

In another embodiment, the present invention provides for maintainingand/or improving insulin sensitivity; and/or treating hyperinsulinemiaand/or insulin resistance in a subject by administering to the subject apharmaceutical composition comprising FRI-1 or a pharmaceuticallyacceptable salt thereof in combination with an anti-diabetic drug,wherein the anti-diabetic drug is selected from the group consisting ofmetformin, sitagliptin and exenatide or a pharmaceutically acceptablesalt thereof. In yet another embodiment, the present invention providesfor decreasing the daily dose of insulin by administering to a subject apharmaceutical composition comprising FRI-1 or a pharmaceuticallyacceptable salt thereof in combination with an anti-diabetic drug,wherein the anti-diabetic drug is selected from the group consisting ofmetformin, sitagliptin and exenatide or a pharmaceutically acceptablesalt thereof.

In yet another embodiment, the present invention provides for treating acondition in a subject comprising administering to the subject a GKactivator in combination with an anti-diabetic drug, wherein thecondition is selected from metabolic disorders (including metabolicsyndrome), glucose intolerance, prediabetic state, insulin resistance,blood glucose lowering, hyperglycemia, impaired glucose tolerance (IGT),Syndrome X, impaired fasting glucose (IFG), type II diabetes, type Idiabetes, delaying IGT to type II diabetes, delaying the progression ofnon-insulin-requiring type II diabetes to insulin-requiring type IIdiabetes, dyslipidemia, hyperlipidemia, hyperlipoproteinemia,hypertension, osteoporosis, non-alcoholic fatty liver disease (NAFLD),complications resulting from or associated with diabetes, (includingnephropathy, retinopathy, neuropathy, impaired wound healing))cardiovascular disease (including arteriosclerosis, atherosclerosis),lowering of food intake, appetite regulation, obesity, regulatingfeeding behavior, and enhancing secretion of enteroincretins.

In yet another embodiment, treatment of type II diabetes includesadministering smaller doses, i.e., less than the optimum dose of eitherGK activator or anti-diabetic drug, or both until a therapeutic effectis attained. In smaller doses, either GK activator or anti-diabetic drugwill offer negligible therapeutic benefits when administered alone. Inyet another embodiment, the GK activator in combination with ananti-diabetic drug is administered simultaneously or sequentially toattain the desired therapeutic effect.

In other embodiments, the present invention provides for methods oftreatment described herein as an adjunct to diet and exercise insubjects with type II diabetes or type I diabetes.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

Examples

Pharmacology

The efficacy of GK activators combined with metformin, sitagliptin orexenatide was examined on different functional end points, e.g., bodyweight or food intake or glucose tolerance or plasma fasting or fedglucose or plasma insulin or insulin sensitivity index in an in vivomodel, e.g., ob/ob mice, relevant to the genetic mechanism over type IIdiabetes.

Fifty male ob/ob mice (approximately 6 weeks of age) were obtained fromCharles River, Italy. Mice were singly housed in polypropylene cageswith free access to a standard diet (Harlan Teklad Global 2018 diet) andtap water at all times. All animals were maintained at 24±2° C. and55±20% humidity on a reverse phase 16 h on/8 h off light-dark cycle(lights on approximately 17:30-9:30 h).

Animals were acclimatized to the animal facility for two weeks. Thefollowing week (i.e., week 3), animals began a daily handling protocol(animals were handled as if to be dosed but were actually not weighed ordosed). Subsequent to the week-long handling protocol, animals weredosed with vehicle orally once daily for a 7 day baseline period (Day −6to 0), i.e., dosing was three weeks after arrival of the animals in thefacility. Dosing began at 08:45 each day so that approximately half ofthe animals were dosed prior to lights off and half after lights off(09:30). Body weight and food and water intake were recorded daily.During the baseline phase (Day −6) animals underwent blood sampling fromthe lateral tail vein (20 μL) with the sample taken into a lithiumheparin-coated tube (Sarstedt CB300LH), i.e., a sample taken from freelyfeeding animals. Blood sampling began at approximately 16:00 to a timedschedule and food was removed immediately after sampling each animal.The following morning, 16 hours after the previous sample, a further(fasted) blood sample (20 μL) was taken from the lateral tail vein intoa lithium heparin-coated collection tube (Sarstedt CB300LH). Food wasreplaced immediately after sampling and the animal was dosed. All bloodsamples were spun in a centrifuge immediately after collection and theplasma fraction stored frozen (−80° C.) prior to determining plasmaglucose (duplicate) and insulin (single replicate) using commerciallyavailable kits and reagents: Alpco mouse ultrasensitive insulin kit80-INSMSU-E10; Thermo Scientific Infinity glucose reagent TR15421.

Subsequently, baseline treatment continued. Towards the end of thebaseline phase, animals were allocated into 6 treatment groups on thebasis of body weight, baseline food and water intake, and fasted plasmaglucose and insulin. Mice were dosed once daily orally (approximately 10weeks of age at the first dose) for 14 days with vehicle or test drug asdetailed in Table 1 below.

TABLE 1 Group Treatment (po; qd) n A Vehicle 8 B FRI-1 (25 mg/kg po) 8 CFRI-1 (75 mg/kg po) 8 D Metformin (100 mg/kg po) 9 E FRI-1 (25 mg/kgpo) + Metformin (100 mg/kg po) 8 F FRI-1 (75 mg/kg po) + Metformin (100mg/kg po) 8

All treatments were dosed orally by gavage once daily. During thetreatment period food intake, water intake and body weight was recordeddaily at the dosing session. At the completion of dosing, animals wereexamined and any overt behavior was recorded. Dosing began atapproximately 08:45 each day for all groups. On Days 6 and 7, bloodsamples were collected as described previously during the baselinephase. Hence, animals underwent blood sampling in the fed state at 16:00on Day 6. Subsequently food was removed from each animal. The followingmorning, a further blood sample was taken (fasted state). This bloodsample was timed such that it was taken 16 hours after the previoussample. Food was returned and the animal was dosed. On each occasion,approximately 20 μL blood was taken into a lithium heparin-coated tube(Sarstedt Microvette CB300LH). Each sample was centrifuged immediatelyand plasma dispensed into an aliquot tube. All plasma samples werefrozen at −80° C. and subsequently assayed for glucose (n=2) and insulin(n=1) content using commercially available kits and reagents: Alpcomouse ultrasensitive insulin kit 80-INSMSU-E10; Thermo ScientificInfinity glucose reagent TR15421.

On day 13, animals underwent blood sampling in the freely fed state (at16:00) as previously described and food was removed subsequent tosampling. On Day 14, the mice underwent an OGTT. Each animal was dosedwith vehicle or test compound and 60 minutes later was dosed withD-glucose (2 g/kg po). Baseline blood samples were taken immediatelyprior to compound dosing (B1) and immediately before the glucose load(B2). Further blood samples were taken 10, 20, 30, 45, 60 and 120minutes post glucose administration. All blood samples (allapproximately 20 μL) were taken from the tail vein. Blood samples weretaken into lithium heparinised tubes (Sarstedt Microvette CB300LH) andplasma separated by centrifugation. All plasma samples were frozen at−80° C. and subsequently assayed for glucose (n=2) and insulin (n=1)content using commercially available kits and reagents: Alpco mouseultrasensitive insulin kit 80-INSMSU-E10; Thermo Scientific Infinityglucose reagent TR15421. Animals were terminated subsequent tocompletion of the OGTT.

Evaluation of exenatide was conducted similar to the evaluation ofmetformin.

Evaluation of FRI-1 with or without sitagliptin was conducted in femalediet induced obese rats. Sixty-eight dietary-induced obese, femaleWistar rats (weight range 250-300 g) were obtained from Charles River(Margate, Kent) and housed in pairs in polypropylene cages with solidfloors and sawdust bedding at a temperature of 21±4° C. and 55±20%humidity. Animals were maintained on a reverse phase light-dark cycle(lights off for 8 h from 09.30 17.30 h) during which time the room wasilluminated by red light. Animals had free access to powdered high fatdiet (VRF1 plus 20% lard), ground chocolate, ground peanuts and tapwater at all times. The three different diets were contained in separateglass feeding jars with aluminum lids (Solmedia Laboratory Suppliers,Romford, Essex). Each lid had a hole cut in it to allow access to thefood. Animals were housed in pairs for at least 14 weeks for theinduction of obesity. Other experimental procedures for rats weresimilar to those for ob/ob mice.

Drugs and Dosing

Animals were dosed with 1% carboxymethylcellulose (Sigma C4888; Lot120M0216V) vehicle for the first 5 days of the baseline phase. For thefinal two days of the baseline phase, animals were dosed with a vehicleof 20% gelucire (Gattefosse; Lot 103201) in distilled water. FRI-1 wasstored refrigerated in a desiccator upon arrival and until use.Metformin was purchased from Sigma (catalog # D150959; Lot BCBF1484V).Exenatide was purchased from American Peptide (catalog #46-3-12B; LotY10049A1). Sitagliptin phosphate was purchased from Tocris Cookson, UK(Lot TCS9133B). The appropriate volume of vehicle (20% gelucire indistilled water) was added directly to the vial containing a weighedamount of drug material. The compound was vortexed for 10 seconds andwas dosed at room temperature. Drug mixtures (i.e., groups E and F) wereadministered as a single dosing bolus. The appropriate volume of vehicle(20% gelucire in distilled water) was added directly to a vialcontaining a weighed amount of both FRI-1 and metformin. The resultingformulation was vortexed for 10 seconds and was dosed at roomtemperature. Drugs were formulated each day immediately prior to dosingand were administered using a dose volume of 2.5 mL/kg. D-Glucose wasadministered in the volume 2.5 mL/kg.

Data Analysis

Body weight and overall and weekly body weight gains (g) were determinedby analysis of covariance with day 1 body weight as a covariate. Foodand cumulative food intake (g) were determined by analysis of covariancewith average daily food intake during the baseline phase (Days −6 to 0)as a covariate. Average food intake was determined using the samemethods. Water intake (g) was determined by a robust regression modelusing M estimation and Huber weighting, using the default parameterc=1.345. The model had treatment as a factor and average daily waterintake during the baseline phase (Days −6 to 0) as covariate. Averagewater intake was analyzed using the same method. Multiple comparisonsagainst the vehicle group were by Williams' test for FRI-1, the multiplet test for Metformin and Dunnett's test for the combination of FRI-1with metformin. The combination of FRI-1 and metformin was compared tometformin alone by Williams' test and to FRI-1 alone by the multiple ttest. Plasma glucose and insulin data were analyzed by robust regressionwith treatment as a factor and bleeding order, baseline body weight,plasma glucose and insulin as covariates, followed by appropriatecomparisons (two-tailed) to determine significant differences from thecontrol group. The statistical methods assume that data are normallydistributed with equal variance in the groups. Initial Shapiro-Wilktests for normality of residuals showed that log(glucose) andlog(insulin) were more normally distributed than glucose and insulin, soa log transformation was used. However, some Shapiro-Wilk tests weresignificant, so robust regression was used to downweight any possibleoutliers in the analysis of these variables.

For the OGTT, the treatment groups were compared at each post-dose timeand by the area under the curves (AUC) for 0-60 minutes and 0-120minutes (AUC_(60min) and AUC_(120min) respectively). These werecalculated as:AUC_(120min)=1/24(2t _(0min)+4t _(10min)+4t _(20min)+5t _(30min)+6t_(45min)+15t _(60min)+12t _(120min))AUC_(60min)=AUC_(120min)−½(t _(60min) +t _(120min))

The AUCs above B2 baselines (AUCB2) were calculated asAUC_(120min)−2t_(0h) and AUC_(60min)−t_(0h). In the above equations, trepresents glucose or insulin concentration at a specific time indicatedby the subscript. A log transformation was used, apart from the AUCBs,which can be negative, so these were not transformed. Analysis of theOGTT was by robust regression model using M estimation, Huber weighting,using the default parameter c=1.345. The model included treatment andassay day as factors and bleeding order, Day 1 body weight and Day −5(fasted) baseline plasma log(glucose) or log(insulin) as covariates. Forthe AUCBs, untransformed Day −5 glucose or insulin was used as acovariate instead of log(glucose) or log(insulin). Fasted plasma glucoseand insulin data on Day 7 were log transformed and analyzed by robustregression with treatment as a factor and bleeding order, Day 1 bodyweight and Day −5 (fasted) baseline plasma log(glucose) or log(insulin)as covariates. Unfasted plasma glucose and insulin data on Day 6 and 13were log transformed and analyzed by robust regression with treatment asa factor and bleeding order, Day 1 body weight and Day −6 (unfasted)baseline plasma log(glucose) or log(insulin) as covariates. Comparisonsagainst vehicle were by Williams' test for FRI-1, the multiple t testfor metformin and Dunnett's test for the combination. The combinationtreatments were compared to metformin by Williams' test and to the samedose of FRI-1 by the multiple t test. P<0.05 was the level accepted forstatistical difference. The chance of a false positive is 5% for eachcompound for each time. Tests were carried out as two-sided tests.

Results of Body Weight Studies with a FRI-1/Metformin Combination

The chronic administration of FRI-1 and metformin showed a synergisticeffect on controlling body weight in male ob/ob mice as shown in FIG. 1.Once daily treatment with the combination of FRI-1 (75 mg/kg po) andmetformin (100 mg/kg po) significantly reduced body weight (p>0.05) onDays 9, 10, 12, 13, and 14 of the study compared to vehicle-treatedcontrols. No other drug treatment had a statistically significant effecton body weight during the dosing phase of the study.

Results of Plasma Insulin Studies with a FRI-1/Metformin Combination

Plasma insulin was determined from freely feeding ob/ob mice. Thechronic administration of FRI-1 and metformin showed a synergisticeffect on increasing insulin sensitivity as shown in FIGS. 2 and 3. Invehicle-treated animals, plasma insulin increased from a baseline levelof approximately 110 ng/mL to 180 ng/mL on Day 13. The high dose FRI-1and metformin combination had a significant effect on plasma insulin.Specifically, this combination treatment significantly reduced plasmainsulin on Day 6 (p>0.05) and Day 13 (p<0.001). This reduction ininsulin was significantly different not only to the levels ofvehicle-treated controls but also to the Day 13 insulin levels ofanimals treated with either 75 mg/kg FRI-1 (p<0.01) or metformin(p<0.001) alone.

Results of Body Weight Studies with a FRI-1/Sitagliptin Combination

The chronic administration of FRI-1 and sitagliptin showed a synergisticeffect on controlling body weight in female diet-induced obese rats asshown in FIG. 4.

Results of Plasma Insulin Studies with a FRI-1/Exenatide Combination

The chronic administration of FRI-1 and exenatide in ob/ob mice showed asynergistic effect on increasing insulin sensitivity as shown in FIGS. 5and 6. Small but significant effects of exenatide and the combination ofexenatide plus FRI-1 (25 mg/kg po) were observed to reduce plasmaglucose on Day 7 when compared to vehicle. There were also significanteffects of exenatide and the combination of exenatide plus both doses ofFRI-1 (25 mg/kg po) to reduce plasma insulin on Day 7 when compared tovehicle. Extra statistical comparisons indicated that the effects ofboth combinations to reduce plasma insulin Day 14 were significantlygreater than the effect of either dose of FRI-1 alone.

What is claimed is:
 1. A method of increasing insulin sensitivity in amammal having type II diabetes comprising administering to the mammal atherapeutically effective amount of a liver-selective glucokinaseactivator in combination with a therapeutically effective amount of aGLP-1 analog or a pharmaceutically acceptable salt thereof, whereininsulin sensitivity in the mammal is increased.
 2. The method of claim1, wherein the mammal is a human.
 3. The method of claim 1, wherein theliver-selective glucokinase activator and the GLP-1 analog areadministered in discrete dosage forms.
 4. The method of claim 1, whereinthe liver-selective glucokinase activator and the GLP-1 analog areadministered in a single dosage form.
 5. The method of claim 1, whereinthe GLP-1 analog is administered by injection and the liver-selectiveglucokinase activator is administered orally.
 6. The method of claim 1,wherein the GLP-1 analog is exenatide or a pharmaceutically acceptablesalt thereof.
 7. A method of improving glycemic control in a mammalhaving impaired glucose metabolism by increasing a mammal's insulinsensitivity comprising administering to the mammal a therapeuticallyeffective amount of a liver-selective glucokinase activator incombination with a therapeutically effective amount of GLP-1 analog or apharmaceutically acceptable salt thereof.
 8. The method of claim 7,wherein the mammal is a human.
 9. The method of claim 7, wherein theliver-selective glucokinase activator and the GLP-1 analog areadministered in discrete dosage forms.
 10. The method of claim 7,wherein the liver-selective glucokinase activator and the GLP-1 analogare administered in a single dosage form.
 11. The method of claim 7,wherein the GLP-1 analog is administered by injection and theliver-selective glucokinase activator is administered orally.
 12. Themethod of claim 7, wherein the GLP-1 analog is exenatide or apharmaceutically acceptable salt thereof.